Note: Descriptions are shown in the official language in which they were submitted.
1
DESCRIPTION
Title of Invention: EXON 18 AND/OR EXON 21 MUTANT EGFR SELECTIVE
INHIBITOR
Technical Field
[0001]
The present invention relates to an antitumor agent against
cancers, comprising an exon 18 and/or exon 21 mutant epidermal
growth factor receptor (hereinafter also referred to as "EGFR").
Background Art
[0002]
EGFR is a receptor-type tyrosine kinase, exerts its
physiological function in noLmal tissue by being bound to
Epidermal Growth Factor (hereinafter also referred to as EGF),
which is a ligand, and contributes to growth and apoptosis
inhibition in epithelial tissues (NPL 1). Further, somatic
mutation of EGFR gene has been known as a cancer-causing gene;
for example, EGFR in which codons 746 to 750 in exon 19 are
deleted (hereinafter also referred to as "exon 19 deletion
mutation") and EGFR in which leucine encoded by codon 858 in exon
21 is mutated to arginine (hereinafter also referred to as "L858R
mutation") constantly induces EGF-independent kinase activity,
and contributes to the growth and survival of cancer cells (NPL
2). These mutations are observed, for example, in 30 to 50% of
non-small-cell lung cancer in East Asia. The mutations are also
observed in about 10% of non-small-cell lung cancer in Europe and
the United States, and are regarded as one of the causes of
cancers (NPL 3).
[0003]
Therefore, research and development of EGFR inhibitor
as an antitumor agent have actively been conducted, and
introduced into the treatment of various EGFR mutation-positive
lung cancers (NPL 2 and NPL 4). Gefitinib, erlotinib, and
afatinib have been used as a therapeutic agent against exon 19
Date Recue/Date Received 2023-12-22
2
deletion mutant and L858R mutant EGFR-positive lung cancers. Exon
19 deletion mutation and L858R mutation account for 90% of EGFR
mutation. Further, occurrence of acquired resistance in the
process of the treatment using these agents has been known, and
50% thereof is caused by resistance mutation EGFR in which codon
790 of exon 20 is changed from threonine to methionine
(hereinafter also referred to as "1790M mutation"). To treat lung
cancers having this mutation, osimertinib has been used as a
therapeutic agent. Therefore, treatments using EGFR inhibitors
are in the process of being established for lung cancer patients
having major EGFR mutations.
[0004]
On the other hand, at present, no treatments using EGFR
inhibitors have been established with respect to some rare EGFR
mutations, such as point mutation or deletion mutation of exon
18, point mutation of exon 21, or the like; and there are reports
that the drug sensitivity of these mutant EGFR varies depending
on the mutation type (NPL 4). For example, the sensitivity of the
lung cancer having point mutation in which glycine encoded by
codon 719 of exon 18 is substituted with an arbitrary amino acid
(hereinafter also referred to as "G719X mutation") or the lung
cancer in which leucine encoded by codon 861 of exon 21 is
substituted with glutamine (hereinafter also referred to as
"L861Q mutation") with respect to gefitinib, erlotinib, and
afatinib is lower than those of exon 19 deletion mutation and
L858R mutation, which are drug-sensitive mutations. Further,
there are reports of skin disorders and digestive tract disorders
as common side effects by the administration of therapeutic doses
of afatinib, gefitinib, and erlotinib. It is widely thought that
these side effects are attributable to inhibition of the function
of wild-type EGFR expressed in normal tissues, such as skin or
digestive tract, by the therapeutic agent (NPL 1); the
development of an inhibitor characterized by lower inhibitory
activity with respect to wild-type EGFR of normal tissues,
compared with mutant EGFR expressed in tumor tissues, has been
Date Recue/Date Received 2023-12-22
3
desired in view of reduction of side effects.
[0005]
Therefore, development of a drug having high inhibitory
activity with respect to exon 18 and exon 21 mutant EGFR, as well
as high selectivity with respect to mutant EGFR compared with
wild-type EGFR, is expected to enable inhibition of growth of
lung cancer cells having mutant EGFR with a dose lower than the
dose causing the side effects in the skin or digestive tract,
thereby contributing to life prolongation or increase in QOL of
patients with mutant EGFR-positive cancers for which treatment
methods have not been established. Further, a drug having high
inhibitory activity with respect to 1790M, which is acquired
resistance mutation against treatments using EGFR inhibitors, is
expected to reduce expression frequency of acquired resistance
during the treatments using EGFR inhibitors against exon 18 or
exon 21 mutant EGFR, which is de novo mutation; and is therefore
expected to contribute to the life prolongation of cancer
patients.
Citation List
Patent Literature
[0006]
PTL 1: W02015/175632A1
PTL 2: W02015/025936A1
Non-Patent Literature
[0007]
NPL 1: Nat. Rev. Cancer, Vol. 6, pp. 803-812 (2006)
NPL 2: Nature Medicine, Vol. 19, pp. 1389-1400 (2013)
NPL 3: Nat. Rev. Cancer, Vol. 7, pp. 169-181 (2007)
NPL 4: Lancet Oncol. Vol. 13, e. 23-31 (2012)
Summary of Invention
Technical Problem
[0008]
Date Recue/Date Received 2023-12-22
4
An object of the present invention is to provide an
antitumor agent that does not cause inhibition of wild-type EGFR
and thus causes smaller side effects, serving as an inhibitor
that can ensure high selectivity with respect to exon 18 and/or
exon 21 mutant EGFR for which the therapeutic effects of the
previously known EGFR inhibitors are insufficient.
Solution to Problem
[0009]
The inventors of the present invention conducted
extensive research, and found that exon 18 and/or exon 21 mutant
EGFR is an appropriate target in treating cancers, and that EGFR
inhibitors conventionally used for the treatments have inferior
selectivity between exon 18 and/or exon 21 mutant EGFR. Further,
the inventors also confirmed that the compound of the present
invention exerts superior selectivity and tumor growth inhibitory
effects with respect to exon 18 and/or exon 21 mutant EGFR. With
this finding, the inventors accomplished the present invention.
[0010]
The present invention encompasses the following
embodiments.
[0011]
Item 1.
An antitumor agent for treating a malignant tumor
patient expressing EGFR having at least one mutation selected
from the group consisting of G719X mutation of exon 18, E709X
mutation of exon 18, and L861X mutation of exon 21, wherein X
represents an arbitrary amino-acid residue, the antitumor agent
comprising (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt
thereof.
[0012]
Item 2.
The antitumor agent according to Item 1, wherein the
exon 18 mutation is at least one mutation selected from the group
Date Recue/Date Received 2023-12-22
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consisting of G719A, G719S, G719C, E709K and E709A.
[0013]
Item 3.
The antitumor agent according to Item 1 or 2, wherein
the exon 21 mutation is L861Q.
[0014]
Item 4.
The antitumor agent according to any one of Items 1 to
3, wherein the EGFR further has T790M mutation.
[0015]
Item 5.
A method for treating a malignant tumor patient,
comprising the step of administering (S)-N-(4-amino-6-methy1-5-
(quinolin-3-y1)-8,9-dihydropyrimido[5,4-b]indolizin-8-
yl)acrylamide or a salt thereof to a malignant tumor patient
expressing EGFR having at least one mutation selected from the
group consisting of G719X mutation of exon 18, E709X mutation of
exon 18, and L861X mutation of exon 21, wherein X represents an
arbitrary amino-acid residue.
[0016]
Item 6.
The method according to Item 5, wherein the exon 18
mutation is at least one mutation selected from the group
consisting of G719A, G719S, G719C, E709K and E709A.
[0017]
Item 7.
The method according to Item 5 or 6, wherein the exon
21 mutation is L861Q.
[0018]
Item 8.
The method according to any one of Items 5 to 7,
wherein the EGFR further has T790M mutation.
[0019]
Item 9.
(S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
Date Recue/Date Received 2023-12-22
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dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
for treating a malignant tumor patient expressing EGFR having at
least one mutation selected from the group consisting of G719X
mutation of exon 18, E709X mutation of exon 18, and L861X
mutation of exon 21, wherein X represents an arbitrary amino-acid
residue.
[0020]
Item 10.
The (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
according to Item 9, wherein the exon 18 mutation is at least one
mutation selected from the group consisting of G719A, G719S,
G719C, E709K and E709A.
[0021]
Item 11.
The (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
according to Item 9 or 10, wherein the exon 21 mutation is L861Q.
[0022]
Item 12.
The (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
according to any one of Items 9 to 11, wherein the EGFR further
has T790M mutation.
[0023]
Item 13.
Use of (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
for treating a malignant tumor patient expressing EGFR having at
least one mutation selected from the group consisting of G719X
mutation of exon 18, E709X mutation of exon 18, and L861X
mutation of exon 21, wherein X represents an arbitrary amino-acid
residue.
[0024]
Item 14.
Date Recue/Date Received 2023-12-22
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The use according to Item 13, wherein the exon 18
mutation is at least one mutation selected from the group
consisting of G719A, G719S, G719C, E709K and E709A.
[0025]
Item 15.
The use according to Item 13 or 14, wherein the exon 21
mutation is L861Q.
[0026]
Item 16.
The use according to any one of Items 13 to 15, wherein
the EGFR further has T790M mutation.
[0027]
Item 17.
Use of (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
for the production of a pharmaceutical agent for treating a
malignant tumor patient expressing EGFR having at least one
mutation selected from the group consisting of G719X mutation of
exon 18, E709X mutation of exon 18, and L861X mutation of exon
21, wherein X represents an arbitrary amino-acid residue.
[0028]
Item 18.
The use according to Item 17, wherein the exon 18
mutation is at least one mutation selected from the group
consisting of G719A, G719S, G719C, E709K and E709A.
[0029]
Item 19.
The use according to Item 17 or 18, wherein the exon 21
mutation is L861Q.
[0030]
Item 20.
The use according to any one of Items 17 to 19, wherein
the EGFR further has T790M mutation.
[0031]
Item 21.
Date Recue/Date Received 2023-12-22
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A pharmaceutical composition comprising (S)-N-(4-amino-
6-methy1-5-(quinolin-3-y1)-8,9-dihydropyrimido[5,4-b]indolizin-8-
yl)acrylamide or a salt thereof and a pharmaceutically acceptable
carrier, for treating a malignant tumor patient expressing EGFR
having at least one mutation selected from the group consisting
of G719X mutation of exon 18, E709X mutation of exon 18, and
L861X mutation of exon 21, wherein X represents an arbitrary
amino-acid residue.
[0032]
Item 22.
The pharmaceutical composition according to Item 21,
wherein the exon 18 mutation is at least one mutation selected
from the group consisting of G719A, G719S, G719C, E709K and
E709A.
[0033]
Item 23.
The pharmaceutical composition according to Item 21 or
22, wherein the exon 21 mutation is L861Q.
[0034]
Item 24.
The pharmaceutical composition according to any one of
Items 21 to 23, wherein the EGFR further has T790M mutation.
[0035]
Item 25.
A method for predicting therapeutic effects of
chemotherapy using an antitumor agent comprising, as an active
ingredient, (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof
in a malignant tumor patient, the method comprising steps (1) and
(2) below:
(1) a step of detecting the presence or absence of
mutation of EGFR gene contained in a biological sample obtained
from the patient; and
(2) a step of predicting that the chemotherapy is
highly likely to exhibit sufficient therapeutic effects with
Date Recue/Date Received 2023-12-22
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respect to the patient when the results of the detection in step
(1) found that the EGFR gene has at least one mutation selected
from the group consisting of G719X mutation of exon 18, E709X
mutation of exon 18, and L861X mutation of exon 21, wherein X
represents an arbitrary amino-acid residue.
[0036]
Item 26.
The method according to Item 25, wherein the exon 18
mutation is at least one mutation selected from the group
consisting of G719A, G719S, G719C, E709K and E709A.
[0037]
Item 27.
The method according to Item 25 or 26, wherein the exon
21 mutation is L861Q.
[0038]
Item 28.
The method according to any one of Items 25 to 27,
wherein the EGFR further has T790M mutation.
[0039]
Item 29.
A method for treating a malignant tumor patient,
comprising steps (1) to (3) below:
(1) a step of detecting the presence or absence of
mutation of EGFR gene contained in a biological sample obtained
from the patient;
(2) a step of predicting that chemotherapy using an
antitumor agent comprising, as an active ingredient, (S)-N-(4-
amino-6-methy1-5-(quinolin-3-y1)-8,9-dihydropyrimido[5,4-
b]indolizin-8-yl)acrylamide or a salt thereof is highly likely to
exhibit sufficient therapeutic effects with respect to the
patient when the results of the detection in step (1) found that
the EGFR gene has at least one mutation selected from the group
consisting of G719X mutation of exon 18, E709X mutation of exon
18, and L861X mutation of exon 21, wherein X represents an
arbitrary amino-acid residue; and
Date Recue/Date Received 2023-12-22
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(3) a step of administering (S)-N-(4-amino-6-methy1-5-
(quinolin-3-y1)-8,9-dihydropyrimido[5,4-b]indolizin-8-
yl)acrylamide or a salt thereof to a malignant tumor patient who
was predicted highly likely to sufficiently respond to the
chemotherapy using an antitumor agent comprising, as an active
ingredient, (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt
thereof, in step (2).
[0040]
Item 30.
The method according to Item 29, wherein the exon 18
mutation is at least one mutation selected from the group
consisting of G719A, G719S, G719C, E709K and E709A.
[0041]
Item 31.
The method according to Item 29 or 30, wherein the exon
21 mutation is L861Q.
[0042]
Item 32.
The method according to any one of Items 29 to 31,
wherein the EGFR further has T790M mutation.
Advantageous Effects of Invention
[0043]
The antitumor agent of the present invention exerts
high selectivity with respect to exon 18 and/or exon 21 mutant
EGFR. Therefore, the antitumor agent of the present invention is
useful in view of providing an antitumor agent that exerts
superior therapeutic effects for a malignant tumor patient
expressing EGFR having exon 18 and/or exon 21 mutation, for which
the therapeutic effects of the previously known EGFR inhibitors
are insufficient.
[0044]
The present invention is also useful in terms of
providing a method for treating a malignant tumor patient
Date Recue/Date Received 2023-12-22
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expressing EGFR having exon 18 and/or exon 21 mutation.
[0045]
The previously known EGFR inhibitors have low
selectivity with respect to exon 18 and exon 21 mutant EGFR,
compared with wild-type EGFR; therefore, the difference between
the dosage for ensuring the antitumor effects and the dosage
causing the side effects (skin disorders, digestive tract
disorders, etc.) derived from wild-type EGFR was small.
Accordingly, the previously known EGFR inhibitors have difficulty
in exerting sufficient therapeutic effects. In contrast, since
the antitumor agent of the present invention has high selectivity
with respect to exon 18 and exon 21 mutant EGFR, it is possible
to increase the dosage without causing side effects derived from
wild-type EGFR. Therefore, the antitumor agent of the present
invention exerts superior therapeutic effects for a malignant
tumor patient expressing EGFR having exon 18 and/or exon 21
mutation.
[0046]
In addition, the antitumor agent of the present
invention exhibited high inhibitory activity with respect to exon
18 and exon 21 mutant EGFR in the presence of T790M mutation,
which is acquired resistance mutation in the exon 20 region.
Therefore, the antitumor agent of the present invention exerts
superior therapeutic effects with respect to a malignant tumor
patient whose response to the existing drug is low due to
acquired resistance mutation caused by the use of existing
antitumor agent.
[0047]
Further, the antitumor agent of the present invention
is also useful in terms of reducing expression frequency of
acquired resistance during the treatments using EGFR inhibitors
against exon 18 or exon 21 mutant EGFR, which is de novo
mutation, because of its high inhibitory activity against exon 18
and exon 21 mutant EGFR even under the presence of T790M
mutation, which is acquired resistance mutation in the exon 20
Date Recue/Date Received 2023-12-22
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region.
Brief Description of Drawings
[0048]
Fig. 1 illustrates the tumor volume (which may be hereinafter
referred to as "TV") of mouse models subcutaneously transplanted
with G719A mutant EGFR-expressing cell line to measure the
antitumor effect of compound A.
Fig. 2 illustrates the body weight during a dosing period of
compounds of mouse models subcutaneously transplanted with G719A
mutant EGFR-expressing cell line to measure the toxicity of
compound A.
Fig. 3 illustrates the tumor volume of mouse models
subcutaneously transplanted with G719A+1790M mutant EGFR-
expressing cell line to measure the antitumor effect of compound
A.
Fig. 4 illustrates the body weight during a dosing period of
compounds of mouse models subcutaneously transplanted with
G719A+1790M mutant EGFR-expressing cell line to measure the
toxicity of compound A.
Description of Embodiments
[0049]
Preferable examples of various definitions in the scope
of the present invention used in this specification are explained
below in detail.
[0050]
In this specification, "EGFR" refers to a human
epidermal growth factor receptor protein, and is also referred to
as ErbB-1 or HER1.
[0051]
In this specification, "wild-type EGFR" refers to EGFR
Date Recue/Date Received 2023-12-22
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free of somatic mutation, which is a protein comprising the amino
acid sequence represented by SEQ ID NO: 1 (GenBank accession
number: NP 005219.2).
[0052]
In this specification, "exon 18" refers to 688-728
region in the amino acid sequence of wild-type EGFR (SEQ ID NO:
1).
[0053]
In this specification, "exon 18 mutation" refers to
point mutation in amino acid in the exon 18 region of wild-type
EGFR (SEQ ID NO: 1). Preferable exon 18 mutation is point
mutation or deletion mutation with 1 amino acid substitution in
the exon 18 region. More preferably, the exon 18 mutation is
E709X, which is point mutation in which glutamic acid encoded by
codon 709 of exon 18 is substituted with an arbitrary amino acid;
or G719X, which is point mutation in which glycine encoded by
codon 719 of exon 18 is substituted with an arbitrary amino acid.
More specifically, preferable examples of E709X include E709K,
which is point mutation in which glutamic acid encoded by codon
709 in the exon 18 region is substituted with lysine; and E709A,
which is point mutation in which glutamic acid encoded by codon
709 in the exon 18 region is substituted with alanine. Preferable
examples of G719X include G719A, which is point mutation in which
glycine encoded by codon 719 in the exon 18 region is substituted
with alanine; G7195, which is point mutation in which glycine
encoded by codon 719 in the exon 18 region is substituted with
serine; and G719C, which is point mutation in which glycine
encoded by codon 719 in the exon 18 region is substituted with
cysteine. Among these, G719A is particularly preferable.
[0054]
In the present invention, "exon 21" refers to 824-875
region in the amino acid sequence of wild-type EGFR (SEQ ID NO:
1).
[0055]
In this specification, "exon 21 mutation" refers to
Date Recue/Date Received 2023-12-22
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point mutation in amino acid in the exon 21 region of wild-type
EGFR (SEQ ID NO: 1). Preferable exon 21 mutation is point
mutation with 1 amino acid substitution in the exon 21 region.
More preferably, the exon 21 mutation is L861X, which is point
mutation in which leucine encoded by codon 861 in the exon 21
region is substituted with an arbitrary amino acid. More
specifically, L861Q, which is point mutation in which leucine
encoded by codon 861 in the exon 21 region is substituted with
glutamine, is preferable.
[0056]
In the present invention, "exon 18 and/or exon 21
mutation" encompasses "exon 18 mutation," "exon 21 mutation," and
"exon 18 and exon 21 mutation."
[0057]
In the present invention, "point mutation" refers to
mutation causing substitution, insertion, or deletion of one or
more (e.g., about 1 to 10, preferably about 1 to 5, more
preferably about 1, 2, or 3) amino-acid residues; and may include
in-frame insertion and/or deletion mutation as nucleic acid.
[0058]
"EGFR having exon 18 and/or exon 21 mutation"
encompasses "EGFR having exon 18," "EGFR having exon 21
mutation," and "EGFR having exon 18 and exon 21 mutation".
[0059]
In this specification, the "EGFR having exon 18
mutation" refers to EGFR having at least one exon 18 mutation.
The EGFR may have two or more different exon 18 mutations, and
preferably has a single exon 18 mutation. Further, the EGFR may
also have a mutation other than exon 18 mutation (such as exon 19
deletion mutation, L858R mutation, or L790M mutation).
[0060]
In this specification, the "EGFR having exon 21
mutation" refers to EGFR having at least one exon 21 mutation.
The EGFR may have two or more different exon 21 mutations, and
preferably has a single exon 21 mutation. Further, the EGFR may
Date Recue/Date Received 2023-12-22
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also have a mutation other than exon 21 mutation (such as exon 19
deletion mutation, L858R mutation, or L790M mutation).
[0061]
Further, EGFR having exon 18 and/or exon 21 mutation
may further have 1790M mutation. 1790M is acquired resistance
mutation in the exon 20 region. 1790M is known to be generated by
the use of existing EGFR inhibitors. The acquisition of T790M
often decreases the effects of existing drug with respect to
malignant tumor patients.
[0062]
In the present invention, specifically, the EGFR having
exon 18 and/or exon 21 mutation further having T790M mutation is
preferably one of EGFR having E709X and/or G719X mutation in the
exon 18 region further having T790M mutation, and EGFR having
L861X mutation in the exon 21 region further having T790M
mutation. In the present invention, more specifically, the EGFR
having exon 18 and/or exon 21 mutation further having T790M
mutation is preferably one of EGFR having E709K or E709A mutation
further having T790M mutation, EGFR having G719A, G719S or G719C
mutation further having T790M mutation, and EGFR having L861Q
mutation further having T790M mutation. Among these, EGFR having
G719A mutation further having T790M mutation and EGFR having
L861Q mutation further having T790M mutation are particularly
preferable.
[0063]
In the present invention, the method for detecting exon
18 and/or exon 21 mutation of EGFR expressed by a malignant tumor
patient is not particularly limited insofar as the method is
capable of detecting the mutations, and any known detection
methods may be used.
[0064]
The sample used in the detection of exon 18 and/or exon
21 mutation is not particularly limited as long as the sample is
a biological sample isolated from a malignant tumor patient, in
particular, a sample obtained from a malignant tumor patient, and
Date Recue/Date Received 2023-12-22
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contains malignant tumor cells. Examples of biological samples
include body fluids (e.g., blood, urine, etc.), tissues, the
extracts thereof, and the cultures of obtained tissues. The
method for obtaining a biological sample can be suitably selected
depending on the type of biological sample.
[0065]
The biological sample is prepared by being
appropriately treated according to the measurement method.
Further, the reagent comprising primer or probe used for the
detection may be prepared by a conventional method according to
the measurement method therefor.
[0066]
In one embodiment of the present invention, a step of
detecting the presence of exon 18 and/or exon 21 mutation of EGFR
expressed by a malignant tumor patient may be performed before
the administration of an antitumor agent to a malignant tumor
patient.
[0067]
A malignant tumor may include two or more different
kinds of malignant tumor cells. Further, two or more malignant
tumors may be generated in a single patient. Therefore, a single
patient may have different mutations in the same amino acid
position of EGFR (for example, the exon 18 mutation is G719A,
G719S and G719C exon 18 mutation; E709K and E709A exon 18
mutation) at the same time.
[0068]
The antitumor agent of the present invention comprises,
as an active ingredient, (S)-N-(4-amino-6-methy1-5-(quinolin-3-
y1)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide (Compound
(A)) or a salt thereof. Compound (A) is represented by the
following chemical formula.
[0069]
Date Recue/Date Received 2023-12-22
17
N
NH ----
2
N
N
0
[0070]
The method for producing the compound of the present
invention is explained below.
Compound A of the present invention may be produced,
for example, through the production method disclosed in
W02015/025936A1, the methods described in the Examples, and the
like. However, the production method of the compound of the
present invention is not limited to these reaction examples.
[0071]
When Compound A of the present invention has isomers
such as optical isomers, stereoisomers, and tautomers, any of the
isomers and mixtures thereof are included within the scope of the
compound of the present invention, unless otherwise specified.
For example, when Compound A of the present invention has optical
isomers, racemic mixtures and the optical isomers separated from
a racemic mixture are also included within the scope of the
compound of the present invention, unless otherwise specified.
[0072]
The salts of Compound A refer to any pharmaceutically
acceptable salts; examples include base addition salts and acid
addition salts.
[0073]
Examples of base addition salts include alkali metal
salts such as sodium salts and potassium salts; alkaline earth
Date Recue/Date Received 2023-12-22
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metal salts such as calcium salts and magnesium salts; ammonium
salts; and organic amine salts such as trimethylamine salts,
triethylamine salts, dicyclohexylamine salts, ethanolamine salts,
diethanolamine salts, triethanolamine salts, procaine salts, and
N,N'-dibenzylethylenediamine salts.
[0074]
Examples of acid addition salts include inorganic acid
salts such as hydrochlorides, sulfates, nitrates, phosphates, and
perchlorates; organic acid salts such as acetates, foLmates,
maleates, fumarates, tartrates, citrates, ascorbates, and
trifluoroacetates; and sulfonates such as methanesulfonates,
isethionates, benzenesulfonates, and p-toluenesulfonates.
[0075]
The compound of the present invention and salts thereof
also encompass prodrugs thereof. A prodrug refers to a compound
that can be converted to the compound of the present invention or
a salt thereof through a reaction with an enzyme, gastric acid,
or the like, under physiological conditions in vivo, i.e., a
compound that can be converted to the compound of the present
invention or a salt thereof by enzymatic oxidation, reduction,
hydrolysis, or the like; or a compound that can be converted to
the compound of the present invention or a salt thereof by
hydrolysis or the like with gastric acid or the like. Further,
the prodrug may be compounds that can be converted to the
compound of the present invention or a salt thereof under
physiological conditions, such as those described in "Iyakuhin no
Kaihatsu [Development of Pharmaceuticals]," Vol. 7, Molecular
Design, published in 1990 by Hirokawa Shoten Co., pp. 163-198.
[0076]
Description of Diseases
Specific examples of tumors targeted in the present
invention include, but are not particularly limited to, head and
neck cancer, gastrointestinal cancer (esophageal cancer, stomach
cancer, duodenal cancer, liver cancer, biliary cancer (e.g.,
gallbladder and bile duct cancer), pancreatic cancer, colorectal
Date Recue/Date Received 2023-12-22
19
cancer (e.g., colon cancer, and rectal cancer), etc.), lung
cancer (e.g., non-small-cell lung cancer, small-cell lung cancer,
and mesothelioma), breast cancer, genital cancer (ovarian cancer,
uterine cancer (e.g., cervical cancer, and endometrial cancer),
etc.), urological cancer (e.g., kidney cancer, bladder cancer,
prostate cancer, and testicular tumor), hematopoietic tumor
(e.g., leukemia, malignant lymphoma, and multiple myeloma),
osteosarcoma, soft-tissue sarcoma, skin cancer, brain tumor, and
the like. Preferable examples include lung cancer, breast cancer,
head and neck cancer, brain tumor, uterine cancer, digestive
organ cancer, hematopoietic tumor, or skin cancer. Lung cancer is
particularly preferable.
[0077]
When Compound A or a salt thereof are used as a
pharmaceutical agent, a pharmaceutical carrier can be added, if
required, thereby forming a suitable dosage form according to
prevention and treatment purposes. Examples of the dosage form
include oral preparations, injections, suppositories, ointments,
patches, and the like. Oral preparations are preferable. Such
dosage forms can be formed by methods conventionally known to
persons skilled in the art.
[0078]
In one embodiment, the antitumor agent of the present
invention is provided as a phaLmaceutical composition comprising
Compound A or a salt thereof, and a pharmaceutically acceptable
carrier.
[0079]
As the pharmaceutically acceptable carrier, various
conventional organic or inorganic carrier materials used as
preparation materials may be blended as an excipient, binder,
disintegrant, lubricant, or colorant in solid preparations; or as
a solvent, solubilizing agent, suspending agent, isotonizing
agent, buffer, or soothing agent in liquid preparations.
Moreover, pharmaceutical preparation additives, such as
antiseptics, antioxidants, colorants, sweeteners, and
Date Recue/Date Received 2023-12-22
20
stabilizers, may also be used, if required.
[0080]
Oral solid preparations are prepared as follows. After
an excipient is added optionally with an excipient, binder,
disintegrant, lubricant, colorant, taste-masking or flavoring
agent, etc., to the compound of the present invention, the
resulting mixture is formulated into tablets, coated tablets,
granules, powders, capsules, or the like by ordinary methods.
[0081]
Examples of excipients include lactose, sucrose, D-
mannitol, glucose, starch, calcium carbonate, kaolin,
microcrystalline cellulose, and silicic acid anhydride. Examples
of binders include water, ethanol, 1-propanol, 2-propanol, simple
syrup, liquid glucose, liquid a-starch, liquid gelatin, D-
mannitol, carboxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac,
calcium phosphate, polyvinylpyrrolidone, and the like. Examples
of disintegrators include dry starch, sodium alginate, powdered
agar, sodium hydrogen carbonate, calcium carbonate, sodium lauryl
sulfate, stearic acid monoglyceride, lactose, and the like.
Examples of lubricants include purified talc, sodium stearate,
magnesium stearate, borax, polyethylene glycol, and the like.
Examples of colorants include titanium oxide, iron oxide, and the
like. Examples of taste-masking or flavoring agents include
sucrose, bitter orange peel, citric acid, tartaric acid, and the
like.
[0082]
When a liquid preparation for oral administration is
prepared, a taste-masking agent, a buffer, a stabilizer, a
flavoring agent, and the like may be added to the compound of the
present invention; and the resulting mixture may be formulated
into an oral liquid preparation, syrup, elixir, etc., according
to an ordinary method.
[0083]
When an injection agent is prepared, a pH regulator, a
Date Recue/Date Received 2023-12-22
21
buffer, a stabilizer, an isotonizing agent, a local anesthetic,
and the like, may be added to the compound of the present
invention; and the mixture may be formulated into a subcutaneous,
intramuscular, or intravenous injection according to an ordinary
method.
[0084]
Examples of the pH adjuster and the buffer used herein
include sodium citrate, sodium acetate, and sodium phosphate.
Examples of the stabilizer include sodium pyrosulfite, EDTA,
thioglycolic acid, and thiolactic acid. Examples of the local
anesthetic include procaine hydrochloride and lidocaine
hydrochloride. Examples of the tonicity agent include sodium
chloride, glucose, D-mannitol, and glycerol.
[0085]
When a suppository is prepared, pharmaceutically
acceptable carriers known in the related field, such as
polyethylene glycol, lanolin, cacao butter, and fatty acid
triglyceride; and as necessary, surfactants such as Tween 80
(registered trademark), may be added to Compound A, and the
resulting mixture may be formulated into a suppository according
to an ordinary method.
[0086]
When an ointment is prepared, a commonly used base,
stabilizer, wetting agent, preservative, and the like, may be
blended into Compound A, as necessary; and the obtained mixture
is mixed and foLmulated into an ointment according to an ordinary
method.
[0087]
Examples of the base include liquid paraffin, white
petrolatum, white beeswax, octyl dodecyl alcohol, and paraffin.
[0088]
Examples of the preservative include methyl
paraoxybenzoate, ethyl paraoxybenzoate, and propyl
paraoxybenzoate.
[0089]
Date Recue/Date Received 2023-12-22
22
When a patch is prepared, the above-described ointment,
cream, gel, paste, or the like, may be applied to an ordinary
substrate according to an ordinary method.
[0090]
Examples of substrates include woven fabrics or non-
woven fabrics comprising cotton, staple fibers, or chemical
fibers; and films or foam sheets of soft vinyl chloride,
polyethylene, polyurethane, etc., may also be used.
[0091]
The amount of Compound A to be incorporated in each of
such dosage unit forms depends on the condition of the patient to
whom the compound is administered, the dosage form thereof, etc.
In general, in the case of an oral agent, the amount of the
compound is preferably 0.05 to 1000 mg per dosage unit form. In
the case of an injection, the amount of the compound is
preferably 0.01 to 500 mg per dosage unit form; and in the case
of a suppository, the amount of the compound is preferably 1 to
1000 mg per dosage unit form.
[0092]
Further, the daily dose of the medicine in such a
dosage form depends on the condition, body weight, age, sex,
etc., of the patient, and cannot be generalized or limited.
Usually, the daily dose for an adult (body weight: 50 kg) of
Compound A may generally be 0.05 to 5000 mg, and preferably 0.1
to 1000 mg; and is preferably administered in one dose, or in two
to three divided doses, per day.
[0093]
The present invention also provides a method for
treating a malignant tumor patient, comprising the step of
administering Compound A or a salt thereof to a malignant tumor
patient expressing EGFR having exon 18 and/or exon 21 mutation.
[0094]
The present invention also provides Compound A or a
salt thereof for treating a malignant tumor patient expressing
EGFR having exon 18 and/or exon 21 mutation.
Date Recue/Date Received 2023-12-22
23
[0095]
The present invention also provides use of Compound A
or a salt thereof for treating a malignant tumor patient
expressing EGFR having exon 18 and/or exon 21 mutation.
[0096]
The present invention also provides use of Compound A
or a salt thereof for the production of a phaLmaceutical agent
for treating a malignant tumor patient expressing EGFR having
exon 18 and/or exon 21 mutation.
[0097]
The present invention also provides a pharmaceutical
composition for treating a malignant tumor patient expressing
EGFR having exon 18 and/or exon 21 mutation, the pharmaceutical
composition comprising Compound A or a salt thereof, and a
pharmaceutically acceptable carrier.
[0098]
The present invention also provides a method for
predicting therapeutic effects of chemotherapy using an antitumor
agent comprising, as an active ingredient, Compound A or a salt
thereof in a malignant tumor patient, the method comprising steps
(1) and (2) below:
(1) a step of detecting the presence or absence of
mutation of EGFR gene contained in a biological sample obtained
from the patient; and
(2) a step of predicting that the chemotherapy is
highly likely to exhibit sufficient therapeutic effects to the
patient when the results of the detection in step (1) found that
the EGFR gene has exon 18 and/or exon 21 mutation.
[0099]
The present invention also provides a method for
treating a malignant tumor patient, comprising steps (1) to (3)
below:
(1) a step of detecting the presence or absence of
mutation of EGFR gene contained in a biological sample obtained
from the patient; and
Date Recue/Date Received 2023-12-22
24
(2) a step of predicting that chemotherapy using an
antitumor agent comprising, as an active ingredient, (S)-N-(4-
amino-6-methy1-5-(quinolin-3-y1)-8,9-dihydropyrimido[5,4-
b]indolizin-8-yl)acrylamide or a salt thereof is highly likely to
exhibit sufficient therapeutic effects with respect to the
patient when the results of the detection in step (1) found that
the EGFR gene has exon 18 and/or exon 21 mutation; and
(3) a step of administering (S)-N-(4-amino-6-methy1-5-
(quinolin-3-y1)-8,9-dihydropyrimido[5,4-b]indolizin-8-
yl)acrylamide or a salt thereof to a malignant tumor patient who
was predicted highly likely to sufficiently respond to the
chemotherapy using an antitumor agent comprising, as an active
ingredient, (S)-N-(4-amino-6-methy1-5-(quinolin-3-y1)-8,9-
dihydropyrimido[5,4-b]indolizin-8-y1)acrylamide or a salt
thereof, in step (2).
[0100]
The base sequence of EGFR gene is publicly known. The
GenBank accession number of the base sequence of cDNA is
NM 005228.4.
[0101]
The "therapeutic effects" can be evaluated by tumor
shrinkage effects, relapse-suppressing effects, life-prolonging
effects, and the like. The relapse-suppressing effects may be
shown as degree of the extension of non-relapse period or the
degree of the improvement in relapse rate; and the life-
prolonging effects may be shown as the degree of the entire
survival time or the degree of the extension of the median of
progression-free survival, or the like. The "sufficient
therapeutic effects" of the chemotherapy using an antitumor agent
comprising, as an active ingredient, Compound A or a salt thereof
means, for example, that superior therapeutic effects are
obtained by the administration of the antitumor agent comprising,
as an active ingredient, Compound A or a salt thereof, such as
extension of survival time, suppression of relapse, and the like,
compared with non-administration.
Date Recue/Date Received 2023-12-22
25
Examples
[0102]
The following describes the present invention in more
detail with reference to the following Test Examples. However,
the present invention is not limited to these Examples (Test
Examples).
[0103]
Test Example 1: In Vitro Drug Efficacy Test
Evaluation Results of Intracellular Phosphorylation in Mutant
EGFR Forced Expression System Using HEK293 Cells (Inhibitory
Activity)
The intracellular target inhibitory activity of
compounds was evaluated based on the following as an index:
intracellular EGFR phosphorylation in a mutant EGFR forced
expression system using Jump-In (trademark) Grip (trademark)
HEK293 cells (TheLmo Fisher Scientific Inc.) (which may be
hereinafter referred to as "HEK293 cells").
HEK293 cells were maintained in D-MEM with GlutaMAX
(trademark)-I (high-glucose) (Thermo Fisher Scientific Inc.) that
contained 10% dialyzed FBS and 100 U/mL penicillin/100 pg/mL
streptomycin (TheLmo Fisher Scientific Inc.); and a pJTITm R4 DEST
CMV pA vector to which a human EGFR gene (G719A, G7195, G719C,
E709K, E709A, L861Q, G719A+T790M, or L861Q+T790M; the symbol "+"
indicates that both mutations are contained) was encoded was
introduced into HEK293 cells, together with Opti-MEM (trademark)
I (Thermo Fisher Scientific Inc.), using a ViaFect (trademark)
Transfection Reagent (Promega Corporation).
HEK293 cells expressing mutant human EGFR were seeded
in each well of a 384-well flat-bottom microplate such that the
cell count per well was 10,000, and incubated in a 5% CO2 gas-
containing incubator at 37 C for 1 day. Compound A, erlotinib,
afatinib, and osimertinib (erlotinib, afatinib, and osimertinib
may be each hereinafter referred to as a "comparative compound")
were individually dissolved in DMSO, and diluted with DMSO or the
Date Recue/Date Received 2023-12-22
26
medium used for suspending the cells. The solutions were then
individually added to each well of the culture plate of the
cells, and the cells were incubated in a 5% CO2 gas-containing
incubator at 37 C for 6 hours. After incubation, the cells were
immobilized using 20% neutral buffered foLmalin (Wako Pure
Chemical Industries, Ltd.), and blocked by an Odyssey (trademark)
blocking buffer (PBS) (M&S TechnoSystems Inc.). The cells were
then reacted with a primary antibody (EGFR Monoclonal Antibody
(R19/48MIX) #AHR5062) (Thermo Fisher Scientific Inc.) that was
diluted with an Odyssey (trademark) blocking buffer (PBS) to
1/200, and a Phospho-EGFR Receptor (Tyr1068) Antibody #2234L
(CST)), followed by subjecting the cells to permeation at 4 C
overnight. The following day, the cells were reacted with a
secondary antibody (IRDye 800CW Goat aRabbit #926-32211 and IRDye
680RD Goat aMouse #926-68070 (M&S TechnoSystems Inc.)) diluted
with an Odyssey (trademark) blocking buffer (PBS) to 1/800, and
subjected to peLmeation at room temperature for 1 hour. The
fluorescence intensity (which may be hereinafter referred to as
"Fl") was detected with an Odyssey Infrared Imaging System (LI-
COR Bioscience) at a fluorescence wavelength of 800 nm and 700
nm.
[0104]
The value obtained by deducting the Fl of a well
without the cells from the Fl detected at a fluorescence
wavelength of 800 nm or 700 nm is referred to as Fl (800, EGFR)-
Blank (for 800 nm) and Fl (700, p-EGFR)-Blank (for 700 nm). The
value obtained by dividing Fl (700, p-EGFR)-Blank of each well by
Fl (800, EGFR)-Blank was determined to be Fl (p-EGFR/EGFR). The
phosphorylated EGFR inhibitory rate was calculated using the
following formula to determine the concentration of the test
compounds at which phosphorylated EGFR was inhibited by 50% (IC5o
(pM)). Table 1 illustrates the results.
[0105]
Phosphorylated EGFR Inhibitory Rate (%) = T/C X 100
T: Fl (p-EGFR/EGFR) of a well to which a test compound was added.
Date Recue/Date Received 2023-12-22
27
C: Fl (p-EGFR/EGFR) of a well to which a test compound was not
added.
[0106]
As is clear from Table 1, compound A exhibited high
inhibitory activity against intracellular phosphorylation of exon
18 or exon 21 mutant EGFR; and the activity was higher than that
of erlotinib or osimertinib, and was equivalent to that of
afatinib. The inhibitory activity of compound A against exon 18
or exon 21 mutant EGFR was higher than that of afatinib in the
presence of T790M mutation, which is an acquired resistant
mutation.
[0107]
Table 1
Compound A of Osimertinib Afatinib Erlotinib
the Present
Invention
G719A 20.5 237.0 20.0 >1000
G719S 22.7 251.9 16.9 539.7
G719C 9.3 102.0 15.3 98.3
E709K 86.0 424.2 14.0 451.4
E709A 103.5 548.9 19.2 478.1
L861Q 40.5 143.1 24.7 608.1
G719A+ 31.3 132.1 110.2 >1000
T790M
L861Q+ 81.8 132.4 177.6 >1000
T790M
[0108]
Test Example 2
Evaluation of Cell Growth Inhibitory Effect Against Wild-Type
EGFR- and Mutant EGFR-Expressing Cell Line (In Vitro)
The inhibitory activity of compounds against wild-type
EGFR and mutant EGFR was evaluated using Ba/F3 cells, which are
mouse B-lymphocyte precursor cell line to which a human EGFR gene
was introduced. Ba/F3 cells were maintained in an RPMI-1640
medium (Thermo Fisher Scientific Inc.) containing 10% fetal
bovine serum (FBS), 100 U/mL penicillin/100 pg/mL streptomycin
(Thermo Fisher Scientific Inc.), and 1 ng/mL mouse interleukin-3
(mIL-3) (CST). A PB-CMV-MCS-EF1-GFP+Puro vector or PB-CMV-MCS-
EF1-RFP+Puro vector into which a human EGFR gene (wild-type (WT),
Date Recue/Date Received 2023-12-22
28
G719A, or L861Q) was encoded was introduced into the cells,
together with a Super PiggyBac Transposase expression vector, by
electroporation using an Amaxa (trademark) Cell Line Nucleofector
(trademark) Kit V, followed by selection using puromycin (SIGMA).
Ba/F3 cells expressing wild-type EGFR (which may be hereinafter
referred to as "Ba/F3¨EGFR WT") exhibited mIL-3-independent
growth in the presence of 50 ng/mL EGF (R&D Systems); and Ba/F3
cells expressing exon 18 or exon 21 active mutant EGFR (which may
be hereinafter referred to as "Ba/F3-EGFR G719A," and "Ba/F3-EGFR
L861Q") exhibited mIL-3-independent growth in the absence of EGF.
[0109]
To evaluate a cell growth inhibitory effect,
Ba/F3¨EGFR WT cells were suspended in an RPMI-1640 medium
containing 10% FBS, 100 U/mL penicillin, 100 pg/mL streptomycin,
and 50 ng/mL EGF; and the cell suspension was seeded in each well
of a 96-well flat-bottom microplate such that the cell count per
well was 30,000. Ba/F3-EGFR G719A cells and Ba/F3-EGFR L861Q
cells were suspended in respective RPMI-1640 mediums containing
10% FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin; and the
cell suspensions were individually seeded in each well of a 96-
well flat-bottom microplate such that the cell count per well was
15,000. Subsequently, compound A, gefitinib, erlotinib, afatinib,
and osimertinib (gefitinib, erlotinib, afatinib, and osimertinib
may be each hereinafter referred to as a "comparative compound")
were individually dissolved in DMSO, and diluted with DMSO or the
medium used for suspending the cells. The solutions were then
added to each well of the culture plate of the cells, and the
cells were incubated in a 5% CO2 gas-containing incubator at 37 C
for 3 days. The cell count after incubation was measured by a
CellTiter-Glo (trademark) Luminescent Cell Viability Assay
(Promega Corporation) in accordance with the manufacturer's
recommended protocol. The growth inhibition rate was calculated
using the following formula to determine the concentration of
each test compound for 50% inhibition (ICm (PM)).
[0110]
Date Recue/Date Received 2023-12-22
29
Growth Inhibitory Rate (%) = T/C x 100
T: the luminescence intensity of a well to which a test compound
was added.
C: the luminescence intensity of a well to which a test compound
was not added.
[0111]
The IC50 ratio between wild-type EGFR and G719A mutant
EGFR, or between wild-type EGFR and L861Q mutant EGFR was
determined using the following formula. Table 2 illustrates the
results.
[0112]
IC50 Ratio = IC50 (WI)/ICm (G719A or L861Q)
[0113]
As is clear from Table 2, compound A exhibited
selective inhibitory activity against G719A mutation and L861Q
mutation.
[0114]
Table 2
IC50 (nM) IC50 Ratio
WT G719A L861Q WT/G719A WT/L861Q
Compound A 597.3 9.0 19.0 66.4 31.4
of the
Present
Invention
Osimertinib 378.7 209.0 47.0 1.8 8.1
Afatinib 17.7 3.3 2.3 5.3 7.6
Erlotinib 778.3 336.3 353.0 2.3 2.2
[0115]
Test Example 3: In Vivo Drug Efficacy Test
Evaluation of Antitumor Effect on Mouse Model Subcutaneously
Transplanted with G719A Mutant EGFR-Expressing Cell Line
The evaluation on mouse models subcutaneously
transplanted with G719A mutant EGFR-expressing cell line was
performed using NIH-3T3 cells, which are mouse fibroblast cell
line to which a human EGFR gene was introduced. NIH-3T3 cells
were maintained in a D-MEM (high-glucose) medium (Wako Pure
Chemical Industries, Ltd.) containing 10% newborn calf serum
Date Recue/Date Received 2023-12-22
30
(NBCS), 1,500 mg/L sodium hydrogen carbonate, and 100 U/mL
penicillin/100 pg/mL streptomycin (TheLmo Fisher Scientific
Inc.); and a PB-CMV-MCS-EF1-RFP+Puro vector into which a human
EGFR gene (G719A) was encoded was introduced to the cells,
together with a Super PiggyBac Transposase expression vector, by
electroporation using an Amaxa (trademark) Cell Line Nucleofector
(trademark) Kit R, followed by selection using puromycin (SIGMA).
NIH-313 cells expressing exon 18 mutant EGFR (which may be
hereinafter referred to as "NIH3T3-EGFR G719A") exhibited growth
in the absence of EGF under 1% NBCS conditions.
[0116]
In evaluation using mouse models subcutaneously
transplanted with G719A mutant EGFR-expressing cell line, nude
mice were subcutaneously transplanted with NIH3T3-EGFR G719A
cells into which a mutant human EGFR was introduced. At the point
at which the tumor volume of the tumor engrafted in the mice grew
to about 100 to 200 mm3, the mice were allocated into groups, 5 or
6 mice for each group, by stratified randomization such that the
average tumor volume between the groups was uniform. The mice
were then orally administered compound A, afatinib, or
osimertinib once daily for 14 consecutive days.
[0117]
The dose of afatinib was 20 mg/kg/day, which is the
maximum tolerated dose (the maximum dose at which the weight loss
during a dosing period is less than 20%), for 14 days, the dosing
period of this test. The dose of osimertinib was 25 mg/kg/day,
which is a clinically efficacious dose. For compound A, three
types of doses were set: 200 mg/kg/day (maximum tolerated dose),
100 mg/kg/day, and 50 mg/kg/day. The maximum tolerated dose was
determined in accordance with the "Guidelines Involving
Experimental Neoplasia Proposals in Mice and Rats" of the
National Cancer Institute (NCI), from a humanitarian perspective.
[0118]
To compare the changes in growth of tumor over time due
to administration of the individual test compounds, the tumor
Date Recue/Date Received 2023-12-22
31
volume (which may be hereinafter referred to as "TV") was used as
an index. For a toxicity index, the body weight was measured over
time, and the body weight change (which may be hereinafter
referred to as "BWC (%)") from the day on which the mice were
divided into groups was calculated in accordance with the
following formula.
[0119]
BWC (%) = (the body weight measured on body weight measurement
day)/(the body weight on the day mice were divided into groups)
[0120]
When the difference in the average TV between the
control group and the group administered with a test compound on
the final evaluation day was statistically significant (Dunnett's
test, p<0.05), and the value of treatment/control (T/C)
calculated using the following formula was less than 100, the
test compound was determined to be effective. Such a case is
indicated by the symbol "k" in the figures.
[0121]
T/C (%) = (the average TV of the group administered with a test
compound)/(the average TV of the control group) x 100
[0122]
As is clear from the results illustrated in Fig. 1,
compound A of the present invention exhibited a remarkable
antitumor effect on G719A mutant EGFR-expressing cell line
subcutaneously transplanted into nude mice, accompanied by tumor
regression. The effect was also higher than that of afatinib or
osimertinib. The mice did not show symptoms, such as serious
weight loss, as illustrated in Fig. 2, abnormal feces, or
abnormal skin.
[0123]
Test Example 4: In Vitro Drug Efficacy Test
Evaluation of Cell Growth Inhibitory Effect on T790M Mutant EGFR-
Expressing Cell Line (In Vitro)
The inhibitory activity of a compound on T790M mutant
EGFR was evaluated using Ba/F3 cells, which are mouse B-
Date Recue/Date Received 2023-12-22
32
lymphocyte precursor cell line into which a human EGFR gene was
introduced. Ba/F3 cells were maintained in an RPMI-1640 medium
(Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum
(FBS), 100 U/mL penicillin/100 pg/mL streptomycin (Thermo Fisher
Scientific Inc.), and 1 ng/mL mouse interleukin-3 (mIL-3) (CST);
and a PB-CMV-MCS-EF1-GFP+Puro vector or PB-CMV-MCS-EF1-RFP+Puro
vector into which a human EGFR gene (wild-type (WT), G719A+1790M,
or L861Q+1790M) was encoded was introduced into the cells,
together with a Super PiggyBac Transposase expression vector, by
electroporation using an Amaxa (trademark) Cell Line Nucleofector
(trademark) Kit V, followed by selection using puromycin (SIGMA).
Ba/F3 cells expressing G719A+1790M mutant EGFR or L861Q+1790M
mutant EGFR (which may be hereinafter referred to as "Ba/F3-EGFR
G719A+1790M," and "Ba/F3-EGFR L861Q+1790M") exhibited mIL-3-
independent growth in the absence of EGF.
[0124]
To evaluate a cell growth inhibitory effect, Ba/F3-EGFR
G719A+T790M cells and Ba/F3-EGFR L861Q+T790M cells were
individually suspended in an RPMI-1640 medium containing 10% FBS,
100 U/mL penicillin, and 100 pg/mL streptomycin; and the cell
suspensions were individually seeded in each well of a 96-well
flat-bottom microplate such that the cell count per well was
15,000. Subsequently, compound A, gefitinib, erlotinib, afatinib,
and osimertinib (gefitinib, erlotinib, afatinib, and osimertinib
may be each hereinafter referred to as a "comparative compound")
were individually dissolved in DMSO, and diluted with DMSO or the
medium used for suspending the cells. These solutions were then
added to each well of the culture plate of the cells, and
incubated in a 5% CO2 gas-containing incubator at 37 C for 3 days.
The cell count after incubation was measured using a CellTiter-
Glo (trademark) Luminescent Cell Viability Assay (Promega
Corporation), in accordance with the manufacturer's recommended
protocol. The growth inhibition rate was calculated using the
following formula to determine the concentration of each test
compound for 50% inhibition (IC5o (11M)).
Date Recue/Date Received 2023-12-22
33
[0125]
Growth Inhibition Rate (%) = T/C x 100
T: the luminescence intensity of a well to which a test compound
was added.
C: the luminescence intensity of a well to which the test
compound was not added.
[0126]
Additionally, the 'Cm ratio between wild-type EGFR and
G719A+1790M mutant EGFR, or between wild-type EGFR and
L861Q+1790M mutant EGFR was calculated using the following
formula. Table 3 illustrates the results.
[0127]
'Cm Ratio = 'Cm (WI)/ICm (G719A+T790M or L861Q+T790M)
[0128]
As is clear from Table 3, compound A exhibited
selective inhibitory activity against G719A+T790M mutation and
L861Q+T790M mutation.
[0129]
Table 3
IC50 (nM) IC50 Ratio
G719A+T790M L861Q+T790M WT/ WT/
G719A+T790M L861Q+T790M
Compound A of 15.8 37.5 37.9 15.9
the Present
Invention
Osimertinib 89.4 36.4 4.2 10.4
Afatinib 68.2 168.7 0.3 0.1
Erlotinib 8030.0 6424.2 0.1 0.1
[0130]
Test Example 5: In Vivo Drug Efficacy Test
Evaluation of Antitumor Effect on Mouse Model Subcutaneously
Transplanted with G719A+T790M Mutant EGFR-Expressing Cell Line
The evaluation of mouse models subcutaneously
transplanted with G719A+T790M mutant EGFR-expressing cell line
was performed using NIH-3T3 cells, which are mouse fibroblast
cell line into which a human EGFR gene was introduced. NIH-3T3
cells were maintained in a D-MEM (high-glucose) medium (Wako Pure
Date Recue/Date Received 2023-12-22
34
Chemical Industries, Ltd.) containing 10% newborn calf serum
(NBCS), 1,500 mg/L sodium hydrogen carbonate, and 100 U/mL
penicillin/100 pg/mL streptomycin (TheLmo Fisher Scientific
Inc.); and a PB-CMV-MCS-EF1-RFP+Puro vector into which a human
EGFR gene (G719A+1790M) was encoded was introduced to the cells,
together with a Super PiggyBac Transposase expression vector, by
electroporation using an Amaxa (trademark) Cell Line Nucleofector
(trademark) Kit R, followed by selection using puromycin (SIGMA).
NIH-313 cells expressing exon 18 mutant EGFR (which may be
hereinafter referred to as "NIH3T3-EGFR G719A+1790M") exhibited
growth in the absence of EGF under 1% NBCS conditions.
[0131]
In evaluation using mouse models subcutaneously
transplanted with G719A+1790M mutant EGFR-expressing cell line,
nude mice were subcutaneously transplanted with NIH3T3-EGFR
G719A+1790M cells into which mutant human EGFR was introduced. At
the point at which the tumor volume of the tumor engrafted in the
mice grew to about 100 to 300 mm3, the mice were allocated into
groups, 5 mice for each group, by stratified randomization such
that the average tumor volume between the groups was uniform. The
mice were then orally administered compound A of the present
invention or afatinib once daily on consecutive days.
[0132]
The dose of afatinib was 20 mg/kg/day, which is the
maximum tolerated dose (the maximum dose at which the weight loss
during a dosing period is less than 20%), for 14 days, the dosing
period of this test. For compound A of the present invention,
three types of doses were set: 200 mg/kg/day (maximum tolerated
dose), 100 mg/kg/day, and 50 mg/kg/day. The maximum tolerated
dose was determined in accordance with the "Guidelines Involving
Experimental Neoplasia Proposals in Mice and Rats" of the
National Cancer Institute (NCI), from a humanitarian perspective.
[0133]
To compare the changes in growth of tumor over time due
to administration of the individual test compounds, the tumor
Date Recue/Date Received 2023-12-22
35
volume (which may be hereinafter referred to as "TV") was used as
an index. Fig. 3 illustrates changes in TV over time. For a
toxicity index, the body weight was measured over time, and the
body weight change (which may be hereinafter referred to as "BWC
(%)") from the day on which the mice were divided into groups was
calculated in accordance with the following formula. Fig. 4
illustrates changes in body weight over time.
BWC (%) = (the body weight measured on body weight measurement
day)/(the body weight on the day mice were divided into groups)
[0134]
A Dunnett's test was performed using the average TV on
the final evaluation day as an index. When the difference in the
average TV between the control group and the group administered
with a test compound was statistically significant (p<0.05), and
the value of treatment/control (T/C) calculated using the
following formula was less than 100, the test compound was
determined to be effective. Such a case is indicated by the
symbol "*" in Fig. 3 and Table 4 (*: p<0.05, **: p<0.01, ***:
p<0.001). Additionally, when the difference in the average TV
between the group administered with compound A of the present
invention and the group administered with afatinib was
statistically significant (p<0.05), and the T/C value of the
group administered with compound A of the present invention was
smaller than the T/C value of the group administered with
afatinib, compound A was determined to have a higher antitumor
effect than afatinib. Such a case is indicated by the symbol "#"
in Fig. 3 and Table 4 (#: p<0.05,##: p<0.01, ###: p<0.001).
[0135]
T/C (%) = (the average TV of the group administered with a test
compound)/(the average TV of the control group) x 100
[0136]
As is clear from the results illustrated in Fig. 3,
compound A of the present invention exhibited a significant
antitumor effect on G719A+T790M mutant EGFR-expressing cell line
Date Recue/Date Received 2023-12-22
36
subcutaneously transplanted into nude mice. Additionally, as
shown in Table 4, the effect was higher than that of afatinib,
without symptoms such as serious weight loss (as illustrated in
Fig. 4), abnormal feces, or abnoLmal skin in mice.
[0137]
Table 4
Compound Dose T/C (%) p value vs
(mg/kg) Control Afatinib
Control 100.0
Compound A 50 47.7 *** N.S.
of the
Present
Invention
Compound A 100 36.4 *** ##
of the
Present
Invention
Compound A 200 28.0 *** ###
of the
Present
Invention
Afatinib 20 57.4 ***
N.S.: No Significant Difference
***: p<0.001 (Dunnett's test vs Control Group)
##: p<0.01 (Dunnett's test vs Afatinib Group)
###: p<0.001 (Dunnett's test vs Afatinib Group)
Date Recue/Date Received 2023-12-22
